Current management of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome/Systemic Exertion Intolerance Disease (ME/CFS/SEID) relies solely on symptom management to improve quality of life but does not address the underlying mechanisms of disease onset and progression. To provide better insight into the key biological targets involved ME/CFS/SEID presentation, the main objective of this research proposal is to identify male-specific biomarkers and therapeutic targets of ME/CFS/SEID and provide insight into sex-specific disease onset and progression, which will lead to the better therapeutic intervention. This research proposal aims to use Peripheral Blood Mononuclear Cells (PBMC) from patients recruited for our recently NIH funded research (1R01NS090200-01 (PI Fletcher)). These PBMC have been isolated from male ME/CFS/SEID patients and matched healthy controls at three time points - before exercise challenge, at the peak of effort (VO2 max) and four hours after the peak effort. Specific Aims of this proposal include: Specific Aim 1. Identify metabolic pathways and possible regulatory RNAs affected in men with ME/CFS/SEID. Our hypothesis is that unknown transcripts and alternative splicing events regulate changes in expression of genes responsible for inflammatory response, leukocyte migration, and regulation of cell development. We aim to use an RNA-seq approach to identify the abundance of known and novel transcripts and discover new splicing events in PBMC of male patients. This will allow establishing a set of sex-specific ME/CFS/SEID candidate transcripts. We will then validate them using NanoString Technologies' nCounter system and create a panel of transcripts to potentially be used in the diagnosis and management of ME/CFS. Specific Aim 2. To investigate possible mechanisms of transcriptional regulation in men with ME/CFS/SEID. To better understand causes of changes in gene expression in male patients with ME/CFS/SEID, we aim to identify changes in copy number variation (CNV) and genomic DNA methylation. We will use Agilent SurePrint G3 Human Genome CGH+SNP microarrays to identify a set of ME/CFS/SEID sex specific candidate CNVs. We will use Illumina Infinium Human Methylation450 BeadChip microarrays to evaluate differences in the methylation patterns between ME/CFS/SEID patients and healthy controls. Specific Aim 3. Compare affected metabolic pathways and mechanisms of transcriptional regulation in men and women with ME/CFS. We will compare differences between ME/CFS/SEID patients and HC in men with the same differences in women (from the recently funded grant proposal 1 R15 NS087604-01A1, PI Nathanson). Comparisons will be done for the results in gene expression, alternative splicing, patterns on the genomic DNA methylation and CNVs. We expect to find differences in the regulation of genes involved in inflammatory response and immune system processes, particularly in the regulation of cytokines production and activity.